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MicroRNA-433 negatively regulates the expression of thymidylate synthase (TYMS) responsible for 5-fluorouracil sensitivity in HeLa cells.

Gotanda K, Hirota T, Matsumoto N, Ieiri I - BMC Cancer (2013)

Bottom Line: The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01).The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05).The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Thymidylate synthase (TYMS) is an important folate-dependent enzyme in DNA synthesis and an important target for cancer chemotherapy. High TYMS expression levels in tumors are generally associated with resistance to 5-fluorouracil (5-FU). The cause of the variability in TYMS expression is still not fully understood, however, only a small proportion of the TYMS expression can be explained by TYMS genetic polymorphisms. The purpose of this study is to identify novel microRNAs (miRNAs) which regulate the expression of TYMS and to determine whether miRNAs binding to the 3'-untranslated region (UTR) of TYMS mRNA affect the proliferation of HeLa cells treated with 5-FU.

Methods: An in silico search was performed to find potential binding sites of miRNAs in TYMS mRNA. The efficacy of predicted miRNAs at the 3'-UTR of TYMS mRNA was evaluated using a dual-luciferase reporter assay. TYMS mRNA and protein expression in HeLa cells was quantified with real-time RT-PCR and Western blotting, respectively. The effects of miR-433 on cell proliferative activity were determined by WST-8 assay.

Results: The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01). The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05). Furthermore, miR-433 increased inhibition of cell proliferation in HeLa cells treated with 5-FU at over 2.0 μM.

Conclusion: The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells. This is the first report showing that a miRNA regulating TYMS expression has a significant impact on sensitivity to 5-FU treatment.

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Effects of miR-433 on the sensitivity to 5-FU in HeLa cells. (A) HeLa cells were transfected with 3.0 nM of pre-miR-negative control (NC), pre-miR-433 (miR-433) or TYMS siRNA. Cell proliferative activities were determined by WST-8 assay. Each column represents the mean ± S.E. for three independent experiments. (B) 5-FU chemosensitivity in HeLa cells was measured by WST-8 assay. Each point represents the mean ± S.D. for three independent experiments. *P < 0.01, compared with the negative control determined by Dunnett’s multiple comparison tests.
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Figure 5: Effects of miR-433 on the sensitivity to 5-FU in HeLa cells. (A) HeLa cells were transfected with 3.0 nM of pre-miR-negative control (NC), pre-miR-433 (miR-433) or TYMS siRNA. Cell proliferative activities were determined by WST-8 assay. Each column represents the mean ± S.E. for three independent experiments. (B) 5-FU chemosensitivity in HeLa cells was measured by WST-8 assay. Each point represents the mean ± S.D. for three independent experiments. *P < 0.01, compared with the negative control determined by Dunnett’s multiple comparison tests.

Mentions: To evaluate the effect of miR-433 on the sensitivity to 5-FU treatment, we performed WST-8 assays on HeLa cells transiently transfected with the pre-miR-negative control, pre-miR-433 or TYMS siRNA. First, we analyzed the effects of overexpression of miR-433 and knock-down of TYMS on cell proliferation without 5-FU treatment; there was no change in proliferation among the transfected cells (Figure 5A). TYMS siRNA significantly decreased proliferation in cells treated with over 0.5 μM of 5-FU compared to the pre-miR-negative control (Figure 5B). In cells overexpressing miR-433, proliferation was not affected at 5-FU concentrations ranging from 0.1 to 1.0 μM; however, the decrease in cell proliferation was remarkable over 2.0 μM, and comparable with that by TYMS siRNA over 3.0 μM. These results indicate that overexpression of miR-433 sensitizes HeLa cells to 5-FU treatment.


MicroRNA-433 negatively regulates the expression of thymidylate synthase (TYMS) responsible for 5-fluorouracil sensitivity in HeLa cells.

Gotanda K, Hirota T, Matsumoto N, Ieiri I - BMC Cancer (2013)

Effects of miR-433 on the sensitivity to 5-FU in HeLa cells. (A) HeLa cells were transfected with 3.0 nM of pre-miR-negative control (NC), pre-miR-433 (miR-433) or TYMS siRNA. Cell proliferative activities were determined by WST-8 assay. Each column represents the mean ± S.E. for three independent experiments. (B) 5-FU chemosensitivity in HeLa cells was measured by WST-8 assay. Each point represents the mean ± S.D. for three independent experiments. *P < 0.01, compared with the negative control determined by Dunnett’s multiple comparison tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750578&req=5

Figure 5: Effects of miR-433 on the sensitivity to 5-FU in HeLa cells. (A) HeLa cells were transfected with 3.0 nM of pre-miR-negative control (NC), pre-miR-433 (miR-433) or TYMS siRNA. Cell proliferative activities were determined by WST-8 assay. Each column represents the mean ± S.E. for three independent experiments. (B) 5-FU chemosensitivity in HeLa cells was measured by WST-8 assay. Each point represents the mean ± S.D. for three independent experiments. *P < 0.01, compared with the negative control determined by Dunnett’s multiple comparison tests.
Mentions: To evaluate the effect of miR-433 on the sensitivity to 5-FU treatment, we performed WST-8 assays on HeLa cells transiently transfected with the pre-miR-negative control, pre-miR-433 or TYMS siRNA. First, we analyzed the effects of overexpression of miR-433 and knock-down of TYMS on cell proliferation without 5-FU treatment; there was no change in proliferation among the transfected cells (Figure 5A). TYMS siRNA significantly decreased proliferation in cells treated with over 0.5 μM of 5-FU compared to the pre-miR-negative control (Figure 5B). In cells overexpressing miR-433, proliferation was not affected at 5-FU concentrations ranging from 0.1 to 1.0 μM; however, the decrease in cell proliferation was remarkable over 2.0 μM, and comparable with that by TYMS siRNA over 3.0 μM. These results indicate that overexpression of miR-433 sensitizes HeLa cells to 5-FU treatment.

Bottom Line: The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01).The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05).The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Thymidylate synthase (TYMS) is an important folate-dependent enzyme in DNA synthesis and an important target for cancer chemotherapy. High TYMS expression levels in tumors are generally associated with resistance to 5-fluorouracil (5-FU). The cause of the variability in TYMS expression is still not fully understood, however, only a small proportion of the TYMS expression can be explained by TYMS genetic polymorphisms. The purpose of this study is to identify novel microRNAs (miRNAs) which regulate the expression of TYMS and to determine whether miRNAs binding to the 3'-untranslated region (UTR) of TYMS mRNA affect the proliferation of HeLa cells treated with 5-FU.

Methods: An in silico search was performed to find potential binding sites of miRNAs in TYMS mRNA. The efficacy of predicted miRNAs at the 3'-UTR of TYMS mRNA was evaluated using a dual-luciferase reporter assay. TYMS mRNA and protein expression in HeLa cells was quantified with real-time RT-PCR and Western blotting, respectively. The effects of miR-433 on cell proliferative activity were determined by WST-8 assay.

Results: The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3'-UTR of TYMS mRNA (P < 0.01). The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05). Furthermore, miR-433 increased inhibition of cell proliferation in HeLa cells treated with 5-FU at over 2.0 μM.

Conclusion: The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells. This is the first report showing that a miRNA regulating TYMS expression has a significant impact on sensitivity to 5-FU treatment.

Show MeSH
Related in: MedlinePlus